This proposal addresses the urgent need for new cellular models of botulinum neurotoxin (BoNT) intoxication for application in high-throughput screening campaigns. Specifically, we will develop cellular model systems that possess high analytical sensitivity and the ability to non-invasively report on intracellular BoNT activity in ral time. Botulinum neurotoxin (BoNT) is the most toxic protein known to man (LD50 ~ 1 ng/kg), where exposure to the toxin ultimately results in host paralysis and death. Despite the broad clinically utility of this toxin, it also can pose a safety threat if misused in the event of a bioterrorist attack. As a result, much effort has been put into the discovery and development of therapeutic strategies for the treatment of botulism. Traditionally, molecules with anti-BoNT activities have been identified through in vitro screening campaigns using fluorescent peptide substrates, or by using cellular models and low-throughput immunological detection of SNARE protein cleavage. Although these efforts have resulted in leads with good in vitro potency, all have failed when advanced to in vivo testing due to poor pharmacokinetic properties such as low aqueous solubility, high cytotoxicity and low cell permeability. In addition, cellular models that rely on secondary cell lines have been shown to have poor predictive value in lead advancement. These limitations, therefore, dictate the need for new cellular models that faithfully recapitulate the phenotype of BoNT intoxication, are better predictors of in vivo efficacy, and are compatible with existing automation and high-throughput screening procedures. In this proposal, we will take advantage of human stem cell-derived motor neurons that have been demonstrated to exhibit high BoNT sensitivities and represent a feasible approach for expansion to cell numbers required for screening applications. Two different approaches will be used to engineer the cells to express a FRET-based reporter previously shown to report intracellular BoNT activity in real time. In the first approach, cells will be prepared to constitutively express the BoNT reporter that will allow for the direct comparison of BoNT sensitivity between differentiation stages. The second prong to our approach is to develop stem cell-derived motor neurons that express the BoNT reporter only in a specific differentiation stage. Stage-specific expression of the reporter protein will allow monitoring of intracellular BoNT activity in real time as well as enable the evaluation of the efficiency of the differentiatio protocol and provide a means to purify cells of interest. Finally, the potential of the developed cellular assays will be evaluated by screening a set of compounds using high content screening equipment with the aim of identifying new leads with anti-BoNT activities that will subsequently be validated using secondary and tertiary screening methods. Through these studies, we propose that viable cellular systems can be produced that will have broad applicability to BoNT antagonist discovery, providing a needed predictive link between in vitro screening models and in vivo bioassays.